Herpes simplex virus 2 (HSV-2) causes genital herpes and increases the risk of transmission and infection with HIV. Thus a vaccine for HSV-2 would not only reduce the rate of genital herpes, but also might reduce spread of HIV. Several HSV-2 vaccines have been tested in humans for prevention or reduction of genital herpes disease, but none has been licensed for use in humans. We performed a randomized, double blind, placebo-controlled clinical trial of a replication-defective vaccine for HSV2 termed HSV529. This vaccine can infect cells, but not replicate in the cells. We vaccinated three groups of 20 subjects with three doses (at 0, 1, and 6 months) of HSV529 (15 subjects per group) or saline placebo injection (5 subjects per group). The groups were a) subjects who were infected with HSV2 in the past but may or may not have been infected with HSV-1 (HSV2+), (b) subjects who were infected only with HSV1 (HSV1+/HSV2-), and (c) subjects who were not infected with HSV1 or HSV2 (HSV1-/HSV2-). Each subject was followed after vaccination, and safety, the primary endpoint, and immune responses to the vaccine were studied. Eighty-nine percent of vaccine recipients experienced mild to moderate solicited injection site reactions compared with 47% of placebo recipients. Sixty-four percent of vaccine recipients experienced systemic reactions compared with 53% of placebo recipients. Seventy-eight percent of HSV1-/HSV2- vaccine recipients had > 4-fold rises in neutralizing antibody titer after three doses of vaccine, whereas none of the participants in the other serogroups had such responses. HSV2-specific CD4+ T-cell responses were detected in 36% 46%, and 27%, of HSV1-/HSV2-, HSV2+, and HSV1+/HSV2- participants, respectively, one month after the third dose of vaccine, and CD8+ T-cell responses were detected in 14%, 8%, and 18% of participants in each group, respectively. Thus, the HSV529 vaccine was safe and elicited neutralizing antibody and modest CD4+ T-cell responses in HSV seronegative vaccine recipients.